FAX 

To: 6'-& NN (VI <':"1 VI \ \ -, 

Fax: '}..O \ ll'?1,LI~ I 

Date: <J 11"1 II <)/ 

Re: C: \> (VI A. 

From: '7C::D~ S'" ~,,,, t '"" 

fax: 202.564.8251 

Phone: ,\--01..' Sb~ . ~~ S I 

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OCSPP OPPT EAD 

1200 Pennsylvania Ave. NW 

Washington, DC 20460 

202.564.8251 

Pages: 

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r- Urgent I- For Review I- Please Comme - Please Reply - Please Recycle -

nt 
UNITED STATES ENVIRONMENTAL PROTECTION AGENCY 

WASHINGTON D.C., 20460 

VIA FACSIMILE: 201-437-4801 

April 19,2012 

Attention: Glenn Merritt (201-437-4000) 

OFFICE OF 

PREVENTION, PESTICIDES AND 

TOXIC SUBSTANCES 

Subject: Follow-up to fax transmission of 4/13/12 (ePMA removal ofCBI claims regarding 

identified studies) 

Dear Mr. Merritt: 

This is sent to you pursuant our conversation of several minutes ago. In Larry Robinson's fax of 

April 13th while there is a waiver of confidential business information (CBI) claims in the 

identified studies there is a an assertion of copyright protections and some additional text that 

provides in part ... "Reproduction, reference, summarization, retransmission or other use of this 

copyrighted document. .. including electronic transmission, using the internet or email .... is 

strictly prohibited .... " 

While we understand the basis for the copyright protections, we have a concern that the language 

in the fax might be interpreted to mean that ePMA is asserting that the studies submitted per the 

TSCA activity are not to be made available in an Agency e-docket (electronic docket) or be 

included in EPA websites among the collection of TSCA health and safety studies. 

We are thinking that the identified limitations noted in the fax are not intended to apply to EPA's 

normal procedures for the handling of non-CBI materials submitted by entities subject to TSCA 

regulations (including the posting of such materials on government websites) and submitted by 

such entities in order to comply with a TSCA requirement, but are rather intended to limit 

entities outside the government from the unauthorized use or appropriation of the studies. 

Can you please confirm CPMA's intent? Many thanks. 

Scott M. Sherlock, Attorney Advisor 

Environmental Assistance Division 

Office of Pollution Prevention and Toxics 


BASF

en-jk 034092-RPT.doc

STUDY TITLE

REPORT

SALMONELLA TYPHIMURIUM / ESCHERICHIA COLI

REVERSE MUTATION ASSAY

(STANDARD PLATE TEST AND PREINCUBATION TEST)

WITH

Alkali Blue Dry 61001 Lot 2236308

DATA REQUIREMENT

OECD No. 471

EC 2000/32, B. 13 / 8.14

AUTHORS

Dr. G. Engelhardt (Study Director)

Dr. E. Leibold

STUDY COMPLETED ON

March 23, 2004

TESTING FACILITY

Experimental Toxicology and Ecology

BASF Aktiengesellschaft

67056 Ludwigshafen, Germany

LABORATORY PROJECT IDENTIFICATION

Project No.: 40M0437/034093

SPONSOR

BASF Akiengeselischaft

67056 Ludwigshafen, Germany IThis docurnent contains manufacturing and trade secrets o1 BASF. it as the property of BASF and may be used only for that purpose for

which it was intended by BASF. Every other or additional use, exploitation, reproduction, publication or submission to other parties

require the written permission of BASF, with the exception of regulatory agencies acting within the lmits of their administrative

authority.

Page 1 of 57
Report; Project No.: 40M0437/034093

THIS PAGE IS INTENTIONALLY LEFT BLANK

Page 2 of 57
BASF

Report, Project No.: 40M0437/034093

GLP COMPLIANCE STATEMENT

This study was conducted in general accordance With the OECD Principles of Good

Laboratory Practice and the GLP Principles of the German "Chemikaliengesete

(Chemicals Act) and meet the United States Environmental Protection Agency Good

Laboratory Practice Standards [40 CFR Part 160 (FIFRA) and Part 792 CrSCA)), with the

exception that recognized differences exist between the GIP Principles/Standards of

OECD and the Principles/Standards of FIFRA and TSCA. However, there was the

following deviation from the requirements of the above-mentioned principles:

-The stability of the test substance in the vehicle DMVSO and in water was not

determined analytically.

Dr. rer. nat. G. Engelhardt

(Study Director)

Page 3 of 57
BASF

Report; Project No.: 40M04371034093

THIS PAGE IS INTENTIONALLY LEFT BLANK

Page 4 of 57
Y~~ Aua I&

Report Project No.: 40M04371034093

SIGNATURE PAGE

-Study Director IConduct of study: 22fra 2a'o C

Dr. rer. nat. G. Engelhardt

Management Aw.~' zd~ oof

Dr. rer. nat E. Leibod

Page 5 of 57
Report; Project No.: 40M0437/034093

STATEMENT

of the Quality Assumnce Unit

The Quality Assurance Unit (QAU) inspected the study and reported any inspection

results to the Study Director and to Management.

The final report reflects the raw data.

Phase of study Date of inspection Reported to Study Directoir

anid to Management

1[mm-dd-yyyyj ['Fmui-dyyyyj

Sdy Plan: 10-20-2003 10-20-2003

Conduct of Study: 12-04-2003 12-04-2003

Report: 03-18-2004 03-18-2004

Ludwigshafenl, 22 - ........ ..... . ......

Page 6 of 57
Report; Project No.: 40M0437/034093

LRhdnlandpfaV

ffir Pflaiztnben mad Pflanzenwacbt

GLPD-Beschelnlgung / Statement of GLP Compliance

(gwnamuI oorft to I1 lb Abe. I Chwnkcflkflauat)

Fine GLP-Inapakdon zur Oboiwachunq der Einhaitung Paissmant of confonnit with GLP according

der GLP-Grundsilzo geniAS Chemikalainoet bzw. to Chemikalengesetz and Directive 85J20/EEC at

Richilrhe 8'20/EG wurdo durcge~ht i:

-jTPrOfelnrlctun Tea flt E) PrOfsandot /Test at

BASF Aktiengesellschaft

Experimentelle Toxikologie und Okologle

0467058 Ludwigshafen

Pr(Ifungen nach Katogorien /Areas of Experli InMftara~fl Ch"VmVWL Mr. 5.SIOECD riowi)

Datum dor Inspoiction /Date of Inspection

......................... .... 5M2001 und vorn 21. bis 28.08.2001......

DIol~or garni ProfenrictuntPr&%tandjort bofindat The above mentioned ba llytest m ~ ite Is hIduded

aidi Imn natlonalen GL-P-Oberwachungsverfahren urd in the national GLP-Caiiplance Prograiwno and Is

iulrd roglnilfig auf Elnhaltig der GLP-GnmdKsttze Inspected onea regular basi.

Auf der Grundiage d83 Inapekllonsberlites wWr Mor- Based on the Irapedon report It can be cordimd,

mit beatfitigt. doss In dowse Pr~evk~uglemem that W3 tout fedWttat ot Is able tW Conduct the

Pr~fatandotd ie oben genmnntan Pro*Jf~ngen tar Eln- aforementioned studies I comipilance with the Pikihalfig doe GLP-Grnamtzs durdgelhrt warden ciples of GLP.

I~rnnf.

Untorschiilt Daw/ln ate

LandO&IW2IaOrPtAe,2enbaI WWi WoIWa~b EasW*&~.M 144, 045128 MOWT

Page 7 of 57
BASF

Report; Project No.: 40M0437/034093

CONTENTS

TITLE PAGE

BLANK PAGE

GLP COMPLIANCE STATEMENT

BLANK PAGE

SIGNATURE PAGE

STATEMENT OF THE QUALITY ASSURANCE UNIT

STATEMENT OF GLP COMPLIANCE (FROM THE COMPETENT AUTHORITY) CONTENTS

1. SUMMARY

2. INTRODUCTION

2.1. PRINCIPLE

2.2. REGULATORY GUIDELINES

3. MATERIAL AND METHODS

3.1. TEST SUBSTANCE

3.2. TEST SUBSTANCE ANALYSIS

3.2.1. Test substance

3.2.2. Test substance preparation

3.3. EXOGENOUS METABOLIC ACTIVATION

3.3.1. S-9 fraction

3.3.2. S-9 mix

3.4. BACTERIA

3.4.1. Salmonella typhimurium

3.4.2. Esoherichia coli

3.4.3. Checking out the tester strains

3.5. EXPERIMENTAL PROCEDURE

3.5-1. Choice of the vehicle

3.5.2. Mutagenicity tests

3.5.2.1. Standard plate test

3.5.2.2. Preincubatlon Test

3.5.3. Titer determination

3.5.4. Control articles

3.5.4.1. Negative controls

3.5.4.2. Positive controls

3.5.5. Scope of tests and test conditions

Page 8 of57
BAS.

Report; Project No.: 40M0437/034093

3.6. EVALUATION

3.6.1. Mutagenicit

3.6.2. Titer

3.6.3. Toxicity

3.6.4. Solubility

3.6.5. Acceptance criteria

3.6.6. Evaluation criteria

3.7. RETENTION OF RECORDS

4. RESULTS

4.1. MUTAGENICITY

4.1.1. Standard plate test

4.1.1.1. Tests without S-9 mix

4.1.1.2. Tests with S-9 mix

4.1.2. Preincubation test

4.1.2.1. Tests without S-9 mix

4.1.2.2. Tests with S-9 mix

4.2. TOXICITY

4.3. SOLUBILITY

4.4. DISCUSSION

4.5. CONCLUSION

5. REFERENCES

Appendix 1: Tables 1 - 9 (Standard plate test)

Appendix 2: Tables 10 - 14 (Preincubation test)

Appendix 3: Sterility control

Appendix 4: S-9 fraction characterization

Appendix 5: Historical negative control data

Appendix 6: Historical positive control data

Page 9 of 57
BASF

Report; Project No.: 40M04371034093

1. SUMMARY

The substance Alkali Blue Dry 6100 / Lot 2236308 was tested for its mutagenic

potential based on the ability to induce point mutations in selected loci of several

bacterial strains, i.e. Salmonella typhimurium and Esoherichia col, in a reverse mutation

assay.

" STRAINS: TA 1535, TA 100, TA 1537, TA 98 and E. coil WP2 uvrA

" DOSE RANGE: 0.8 pg - 5 750 pg/plate (SPT; Salmonella strains)

23.0 pg - 5 750 pg/plate (SPT; E. coli WP2 uvrA)

4.0 pg - 1 500 pg/plate (PIT; all tester strains)

*TEST CONDITIONS: Standard plate test (SPT) and preincubation test (PIT) both

with and without metabolic activation (Aroclor-induced rat liver

S-9 mix).I

*SOLUBILITY: Precipitation of the test substance was found from about

100 pg/plate onward.

*TOXICITY: A bacteriotoxic effect was observed under all test conditions

(for details see item 4.2.).

*MUTAGENICITY:

An increase in the number of his* or trp4 revertants was not observed in the standard

plate test or in the preincubation test either without S-9 mix or after the addition of a

metabolizing system.

*CONCLUSION:

According to the results of the present study, the test substance

Alkali Blue Dry 6100 / Lot 2236308 is not mutagenic in the Salmonella

typhimurium/Escherichis coli reverse mutation assay under the experimental

conditions chosen here.

Page 10 of 57
Report; Project No.: 40M0437/034093

2. INTRODUCTION

2.1. PRINCIPLE

Bacteria[ reverse mutation assays using amino-acid requiring strains of Salmonella

typhimuriumn and Escherichia coi are commonly employed as initial screening methods

for detecting a point mutagenic activity of chemical substances (1, 2, 5) and are used to

screen for possible mammalian mutagens and carcinogens.

The principle of these assays is that mutations are detected by the reversion of mutations

present in the bacterial strains. This leads to a restoration of the functional capability of

the bacteria to synthesize the essential amino acid and thus to the ability to grow in the

absence of the amino acid required by the parent strains.

Most of the substances are not mutagenic or carcinogenic themselves, but only after

metabolic transformation, which in vivo is catalyzed mainly by the enzyme systems of the

liver. Therefore, the tests are carried out not only directly, but also in the presence of an

exogenous metabolic activation system. The most commonly used system is a cofactorsupplemented postmitochondrial fraction (S-9) obtained from livers of rats treated with an

enzyme-inducing agent, e.g. Aroclor 1254.

Experimental starting date: November 6, 2003

Experimental completion date December 12, 2003

(evaluation last experiment):

2.2. REGULATORY GUIDELINES

The tests are carried out in accordance with the following guidelines:

e OECD No. 471 (July 21, 1997)

e EC Directive 2000/32, B-13 IB.14 (May 19, 2000)

Page 11 of 57
- - i ~ra~ ~imI IJULIII~u~ui~I~ilL I JLIIIL II L L i IL

BASF

Report; Project No.: 40M0437/034093

3. MATERIAL AND METHODS

3.1. TEST SUBSTANCE

Name of test substance: Alkali Blue Dry 6100 /Lot 2236308

Test substance No.: 03/0437-3

Batch No.: Lot 2236308

GAS No.: 1324-76-1

Date of filling: September 18, 2003

Purity/Composition: 88 % main component

(See analytical data of October 9, 2003)

Appearance, consistency: Blue Powder

Storage: Room temperature

Page 12 of 57
BASF

Report; Project No.: 40M0437/034093

3.2. TEST SUBSTANCE ANALYSIS

3.2.1. Test substance

The stability of the test substance under storage conditions is guaranteed as indicated by

the sponsor, and the sponsor holds this responsibility.

The homogeneity of the test substance was guaranteed by mixing before preparation of

the test substance formulations.

The analytical investigations were carried out in the:

-Analytical Laboratory of BASF Corporation Huntington (characterization)

3.2.2. Test substance preparation

The stability of the test substance in the vehicle DMSO and in water was not determined

analytically.

More detailed information about the test substance can be found in the raw data and may

be requested from the sponsor (BASF Aktiengesellschaft).

Page 13 of 57
BASF

Report; Project No.: 40M0437/034093

3.3. EXOGENOUS METABOLIC ACTIVATION

3.3.1. S-9 fraction

The S-9 fraction is prepared according to Ames et al. (1, 2).

At least 5 male Sprague-Dawley rats (200 - 300 g, Charles River Deutschland GrnbH) receive a

single intraperitoneal injection of 500 mg Aroclor 1254 (as a solution in corn oil with a

concentration of 20 g/l 00 ml) per kg body weight 5 days before sacrifice.

During this time, the animals are housed in Makmolon cages in air-conditioned rooms in which

central air conditioning guaranteed a range of temperature of 20 - 24C and a relative humidity of

30 - 70%. The day/night rhythm is 12 hours (light period from 6.00 - 18.00 hours and dark period

from 18.00 -6.00 hours).

Standardized pelleted feed and tap water from bottles are available ad libitum.

5 days after administration, the rats are sacrificed, and the livers are prepared (all preparation

steps for obtaining the liver microsome enzymes are carried out using sterile solvents and

glassware at a temperature of +4*C). The livers are weighed and washed In an equivalent

volume of a 150 mM KCI solution, then cut into small pieces and homogenized in three volumes

of KCI solution. After centifugation of the homogenate at 9 000 x g for 10 minutes at +40C, 5 ml

portions of the supernatant (so-called S-9 fraction) are stored at -700 to -80*C.

The preparation date and protein content of the S-9 fraction are given in Appendix 4.

3.3.2. S-9 mix

The S-9 mix is prepared freshly prior to each experiment (1, 2). For this purpose, a sufficient

amount of S-9 fraction is thawed at room temperature and 1 volume of S-9 fraction Is mixed with

9 volumes of S-9 supplement (cofactors). This preparation, the so-called S-9 mix, is kept on ice

until used. The concentrations of the cofactors In the S-9 mix are:

MgC 2 8 mM

KCI 33 mM

Glucose-6-phosphatase 5 mM

NADP 4 mM

Phosphate buffer (pH 7.4) 15 mM

The phosphate buffer (6) is prepared by mixing an Na2HPOA solution with an NaH2PO4 solution in

a ratio of about 4 :1.

To demonstrate the efficacy of the S-9 mix in this assay, the S-9 batch was characterized with

benzo(a)pyrene (see Appendix 4).

Page 14 of 57
Report; Project No.: 40M04371034093

3.4. BACTERIA

For testing, deep-frozen (-700C to -800C) bacterial cultures (Salmonella typhimurium

TA 1535, TA 100, TA 1537, TA 98 and E. coi WP2 uvrA) are thawed at room

temperature, and 0.1 ml of this bacterial suspension is inoculated in nutrient broth

solution (8 g Difco nutrient broth + 5 g NaCI/liter) and incubated in the shaking water bath

at 37*C for about 10 - 12 hours. As a rule, a germ density of > 108 bacteria/ml is reached.

These cultures grown overnight are kept in iced water from the beginning of the

experiment until the end in order to prevent further growth.

The use of the strains mentioned is in accordance with the current scientific

recommendations for the conduct of this assay.

The Salmonella strains were obtained from KNOLL Aktiengesellschaft on October 30,

1989. The Escherichia coli strain was obtained from Merck on September 9, 1991.

3.4.1. Salmonella typhimurium

The rate of induced back mutations of several bacteria mutants from histidine auxotrophy

(his-) to histidine prototrophy (his*) is determined (2, 3, 4). The tester strains TA 1535,

TA 1537, TA 98 and TA 100 selected by Ames and coworkers are derivatives of

Salmonella typhimurium LT2 and have GC base pairs at the primary reversion site. All

strains have a defective excision repair system (uvrB), which prevents the repair of

lesions which are induced in the DNA, and this deficiency results in greatly enhanced

sensitivity of some mutagens. Furthermore, all strains show a considerably reduced

hydrophilic polysaccharide layer (rfa), which leads to an increase in permeability to

lipophilic substances.

The strains TA 1535 and TA 100 are derived from histidine-prototrophic Salmonella

strains by the substitution mutation his G 46 and are used to detect base pair

substitutions. TA 1537 and TA 98 are strains for the detection of frameshift mutagens.

These strains carry different frameshift markers, i.e. the +1 mutant his C 3076 in the case

of TA 1537 and the +2 type his D 3052 in the case of TA 98.

The strains TA 98 and TA 100 carry an R factor piasmid pKM 101 (4) and, in addition to

having genes resistant to antibiotics, they have a modified postreplication DNA repair

system, which increases the mutation rate by inducing a defective repair in the DNA; this

again leads to a considerable increase in sensitivity.

Page 15 of 57
BASV

Report; Project No.: 40M04371034093

3.4.2. Escherichia coil

Escherichia col WP2 uvrA which has an AT base pair at the primary reversion site is a

derivative of E. col WP2 with a deficient excision repair and is used to detect substances which induce base pair substitutions (5). The rate of induced back mutations

from tryptophan auxotrophy (tr) to tryptophan independence (tVp) is determined.

3.4.3. Checking out the tester strains

The Salmonella strains are checked for the following characteristics at regular Intervals:

deep rough character (rta); UV sensitivity (,& uvrB); ampicillin resistance (R factor

plasmid).

E. col WP2 uvrA is checked for UV sensitivity.Iw

Histidine and tryptophan auxotrophy is automatically checked in each experiment Via the

spontaneous rate.

Page 16 of 57
Report; Project No.: 40M0437/034093

3.5. EXPERIMENTAL PROCEDURE

3.5.1. Choice of the vehicle

Due to the limited solubility of the test substance in water, DMSO was selected as the

vehicle, which had been demonstrated to be suitable in bacterial reverse mutation tests

and for which historical control data are available.

3.5.2. Mutagenicity tests

3.5.2.1. Standard plate test

Salmonella typhimurium

The experimental procedure of the standard plate test (plate incorporation method) is

based on the method of Ames et al. (1, 2).

Test tubes containing 2-mi portions of soft agar (overlay agar), which consists of

100 ml agar (0.6% agar +- 0.6% NaCI) and 10 ml amino acid solution (minimal amino

acid solution for the determination of mutants: 0.5 mM histidine + 0.5 mM biotin) are

kept in a water bath at 45*C, and the remaining components are added in the following

order:

0.1 ml test solution or vehicle

0.1 ml fresh bacterial culture

0.5 ml S-9 mix (in tests With metabolic activation)

or

0.5 ml phosphate buffer (in tests without metabolic activation)

After mixing, the samples are poured onto Vogel-Banner agar plates (minimal glucose

agar plates) within approx. 30 seconds.

Composition of the minimal glucose agar:

980 ml aqua dest.

20 ml Vogel-Banner E medium

15g Difco bacto agar

20 g D-glucose, monohydrate.

After incubation at 37'C for 48 - 72 hours in the dark, the bacterial colonies

(his" revertants) are counted.

Escherichia coli

Page 17 of 57
BASF

Report; Project No.: 40M0437/034093

The experimental procedure is based on the method of Ames et al. (1, 2).

Test tubes containing 2-mI portions of soft agar (overlay agar), which consists of

100 ml agar (0.6% agar +-0.6% NaCl) and 10 ml amino acid solution (minimal amino

acid solution for the determination of mutants: 0.5 mM tryptophan) are kept in a water

bath at 45TC, and the remaining components are added In the following order

0.1 ml test solution or vehicle

0.1 ml fresh bacterial culture

0.5 ml S-9 mix (in tests with metabolic activation)

or

0.5 ml phosphate buffer (in tests without metabolic activation)

After mixing, the samples are poured onto minimal agar plates within approx.

30 seconds.

Composition of the minimal agar:

The composition of the minimal agar (SAl selective agar) is based on the description

of Green, M.H.L. and Muriel, W.J. (5), wvith the exception of solution E (tryptophan

solution), which has previously been added to the soft agar

300 ml solution B (agar) 100 ml solution A (saline solution)

8 ml solution C (glucose solution)

10 ml solution D (casein solution)

After incubation at 370C for 48 - 72 hours in the dark, the bacterial colonies

(trp* revertants) are counted.

Page 18 of 57
B-RASF

Report; Project No.: 40MO437/034093

3.5.2.2. Preincubation Test

The experimental procedure is based on the method described by Yahagi et al. (7) and

Matsushima et al. (8).

0. 1 ml test solution or vehicle, 0. 1 ml bacterial suspension and 0.5 ml S-9 mix or

phosphate buffer are incubated at 37*C for the duration of about 20 minutes using a

shaker. Subsequently, 2 ml of soft agar is added and, after mixing; the samples are

poured onto the agar plates within approx. 30 seconds.

After incubation at 37'C for 48 - 72 hours in the dark, the bacterial colonies are counted.

3.5.3. Titer determination

In the standard plate test, 0. 1 ml of the overnight cultures is diluted to 10~ in each case.

Test tubes containing 2-mI portions of soft agar containing maximal amino acid solution

(5 mM tryptophan or 5 mM histidine + 0.5 mM biotin) are kept in a water bath at 450C,

and the remaining components are added in the following order:

0.1 ml vehicle (without and with test substance)

0.1 ml fresh bacterial culture (dilution: 1O0)

0.5 ml S-9 mix

In the preincubation test, 0. 1 ml of the overnight cultures is diluted to 10O in each case.

0. 1 ml vehicle (with and without test substance), 0. 1 ml bacterial suspension and 0.5 ml

S-9 mix are incubated at 37*C for about 20 minutes using a shaker. Subsequently, 2 ml

of soft agar containing maximal amino acid solution for titer determination (5 mM

tryptophan or 5 mM histidine + 0.5 mM biotin) is added.

After mixing, the samples are poured onto the agar plates within approx. 30 seconds.

After incubation at 370C for 48 - 72 hours in the dark, the bacterial colonies are counted.

Page 19 of 57
BASF

Report; Project No.: 40M0437/034093

3.5.4. Control articles

3.5.4.1. Negative controls

Parallel with each experiment, negative controls are carried out in order to check for

possible contaminants (sterility control) and to determine the spontaneous mutation rate

(vehicle control).

*Sterility control

Additional plates are treated with soft agar, S-9 mix, buffer, vehicle or the test

substance but without the addition of tester strains (see Appendix 3).

*Vehicle control

The vehicle control with and without S-9 mix only contains the vehicle used for-the test

substance at the same concentration and volume for all tester strains.

3.5.4.2. Positive controls

The following positive controls are used to check the mutability of the bacteria and the

activity of the S-9 mix:

With S-9 mix

*2-aminoanthracene (2-AA) (SIGMA, A-1 381)10

- 2.5 pg/plate, dissolved in DM50

- strains: TA 1535, TA 100, TA 1537, TA 98

- 60 pg/plate, dissolved in DM50

- strain: Escherichia coli WP2 uvrA

Page 20 of 57
BASF

Report; Project No.: 40M0437/034093

Without S-9 mix

*N-methyl-1'-nitro-N-nitrosoguanidine (MVNNG) (FLUKA, 68051) -5 pg/plate, dissolved in DMSO

- strains: TA 1535, TA 100

*4-nitro-o-phenylendiamine (NOPID) (SIGMA, N-9504) - 10 pg/plate, dissolved in DMSO

- strain: TA 98

*9-aminoacridine (AAC) (SIGMA. A-I 135) - 100 pg/plate, dissolved in DMS0

- strain: TA 1537

*4-nitroquinoline-N-oxide (4-NQO) (SIGMA, N-8141)

- 5 pg/plate, dissolved in DMS0

- strain: E. coli WP2 uvrA

The stability of the selected positive controls is well-defined under the selected culture

conditions, since they are well-established reference mutagens.

Page 21 of 57
BASF

Report; Project No.: 40M0437/034093

3.5.5. Scope of tests and test conditions

1st Experiment

Strains: TA 1535, TA 100, TA 1537, TA 98,

E. col WP2 uvrA

Doses: 0; 23; 115; 575: 2 875 and 5 750 pg/plate

Vehicle: DMSO

Type of test: Standard plate test with and without S-9 mix

Number of plates: 3 test plates per dose or per control

2nd Experiment

Strains: TA 1535, TA 100, TA 1537, TA 98,

Doses: 0; 0.8; 4; 20; 100 and 500 pg/plate

Vehicle: DMS0

Type of test: Standard plate test with and without S-9 mix

Number of plates: 3-test plates per dose or per control

3rd Experiment

Strains: TA 1535, TA 100, TA 1537, TA 98,

E. coil WP2 uvrA

Doses: 0; 4; 20;:100; 500 and 1 500 pg/plate

Vehicle: DMSO

Type of test: Preincubation test with and without S-9 mix

Number of plates: 3 test plates per dose or per control

Page 22 of 57
BASF

Report; Project No.: 40M0437/034093

3.6. EVALUATION

3.6.1. Mutagenicity

Individual plate counts, the mean number of revertant colonies per plate and the standard

deviations were given for all dose groups as well as for the positive and negative

(vehicle) controls in all experiments. In general, five doses of the test substance are

tested With a maximum of 5 mg/plate, and triplicate plating is used for all test groups at

least in the 1st experiment. Dose selection and evaluation as well as the number of

plates used in repeat studies or further experiments are based on the findings of the 1st

experiment.

3.6.2. Titer

The titer is generally determined only in the experimental parts with S-9 mix both for the

negative controls (vehicle only) and for the two highest doses in all experiments.,

3.6.3. Toxicity

Toxicity detected by a

- decrease in the number of revertants

- clearing or diminution of the background lawn (= reduced his- or trp background

growth) - reduction in the titer

is recorded for all test groups both with and without S-9 mix in all experiments and

indicated in the tables.

3.6A4. Solublilty

Precipitation of the test material is recorded and indicated in the tables. As long as

precipitation does not interfere with the colony scoring, 5 mg/plate is generally selected

and analyzed (in cases of nontoxic compounds) as the maximum dose at least in the 1st

experiment even in the case of relatively insoluble test compounds to detect possible

mutagenic impurities. Furthermore, doses > 5 mg/plate might also be tested in repeat

experiments for further clarification/substantiation.

Page 23 of 57
fl~III~inLMKjiLL4IIJ ] At-I I

BASS:

Report; Project No.: 40M0437/034093

3.6.5. Acceptance criteria

Generally, the experiment is considered valid if the following criteria are met:

" The number of revertant colonies in the negative controls was within the normal range

of the historical control data for each tester strain (see Appendix 5).

" The sterility controls revealed no indication of bacterial contamination.

" The positive control articles both with and without S-9 mix induced a significant

increase in the number of revertant colonies within the range of the historical control

data or above (see Appendix 6).

*The titer of viable bacteria was > I 081m1.

3.6.6. Evaluation criteria

The test chemical is considered positive in this assay if the following criteria are met

*A dose-related and reproducible increase in the number of revertant colonies, i.e.

about doubling of the spontaneous mutation rate in at least one tester strain either

without S-9 mix or after adding a metabolizing system.

A test substance is generally considered nonmutagenic in this test If:

*The number of revertants.for all tester strains were within the historical negative

control range under all experimental conditions in two experiments caried out

independently of each other.

Page 24 of 57
Report; Project No.: 40M0437/034093

3.7. RETENTION OF RECORDS

GLP-relevarit records and materials are stored at BASF Aktiengesellschaft and at

BASF Corporation at least for the period of time specified in the GLP principles. Details

on responsibilities or locations of archiving may be seen from the relevant SONs and

from the raw data.

Page 25 of 57
BASF

Report; Project No.: 40M0437/034093

4. RESULTS

(Tables 1 - 14)

The substance Alkali Blue Dry 6100 / Lot 2238308 was tested for mutagenicity in the

Salmonella Ityphimurium / Escherichia coi reverse mutation assay both in the standard

plate test and in the preincubation test with and without the addition of a metabolizing

system (S-9 mix) obtained from rat liver using the Salmonella strains TA 1535, TA 100,

TA 1537, TA 98 and Escherichia coil WP2 uvrA.

4.1. MIITAGENICITY

4.1.1. Standard plate test

(Appendix 1, Tables I - 9)

4.1.1.1. Tests without S-9 mix

TA 1535:

TA 100:

TA 1537: No increase in the number of his" or trp4 revertants.

TA 98:

E. col WP2 uvrA:

4.1.1.2. Tests with S-9 mix

TA 1535:

TA 100:

TA 1537: No increase in the number of his+ or trp* revertants.

TA 98:

E. coi WP2 uvrA:

Page 26 of 57
Report; Project No.: 40M0437/034093

4.1.2. Preincubation test

(Appendix 2, Tables 10 - 14)

4.1.2.1. Tests without S-9 mix

TA 1535:

TA 100:

TA 1537: No increase in the number of hise or ti-p' revertants.

TA 98:

E. coi WP2 uvrA:

4.1.2.2. Tests with S-9 mix

TA 1535:

TA 100:

TA 1537: No increase in the number of hie4 or trp4 revertants.

TA 98:

E. coi WP2 uvrA:

Page 27 of 57
RAWF

Report; Project No.: 40M0437/034093

4.2. TOXICITY

A bacteriotocic effect (reduced background growth, decrease in the number of revertants,

reduction in the titer) was observed in the standard plate test using the Salmonella

strains at doses > 575 pg/plate. With E. coli WP2 uvrA only a slight decrease in the

number of trp~ revertants was found at doses ! 2 875 pg/plate.

In the preincubation assay bacteriotoxicity (reduced background growth, decrease in the

number of revertants) was occasionally observed at about 1 500 pg/plate.

4.3. SOLUBILITY

Test substance precipitation was found from about 100 pg/plate onward.

4.4. DISCUSSION

According to the results of the present study, the test substance did not lead to an

increase in the number of revertant colonies either without S-9 mix or after adding a

metabolizing system in three experiments carried out independently of each other

(standard plate test and preincubation assay).

4.5. CONCLUSION

Thus, under the experimental conditions chosen here, it is concluded that

Alkali Blue Dry 6100 ILot 2236308 is not a mutagenic agent in a bacterial reverse

mutation test.

Page 28 of 57
Report; Project No.: 40M0437/034093

&.REFERENCES

1 . Ames, B.N.; McCann, J.; Yamasaki, E.:

Methods for detecting carcinogens and mutagens with the

Salmonella/mammalian-microsome mutagenicity test.

Mut. Res., 31,. 347 - 364 (1975)

2. Maron, D.M.; Ames, B.N.:

Revised method for the Salmonella mutagenicity test.

Mut. Res., 113,217 -233 (1983)

3. Ames, B.N.; Lee, F.D.; Durston, W.E.:

An improved bacterial test system for the detection and classification of mutagens

and carcinogens.

Proc. Nat. Acad. Sci. USA, 7, 782 -786 (1973)

4. McCann, J.; Spingam, N.E.; Kobori, J.; Ames, B.N.:

Detection of carcinogens as mutagens: Bacterial tester strains with R factor plasmids.

Proc. Nat. Acad. Sci. USA, 72, 979 - 983 (1975)

5. Green, M.H.L;, Muriel, W.J.:

Mutagen testing using trp* reversions in Escherichia coll.

Mut Res., 38, 3 - 32 (1976)

6. DeMarini, D.M.; Dallas, M.M.; Lewias, J.:

Cytotoxicity and effect on mutagenicity of buffers In a microsuspension assay.

Ter. Carc. Mut., 9, 287 - 295 (1989)

7. Yahag i, T.; Nagao, M.; Seino, Y.; Matsushima, T.; Sugimura, T.; Okada, M.:

Mutagenicities of N-nitrosoamines in Salmonella.

Mut Res.,A8, 121 -130 (1977)

8. Matsushimna, T.; Sugimura, T.; Nagao, M.; Yahagi, T.; Shirai, A.:

Factors modulating mutagenicity in microbial tests.

in: Norpoth, K.H. and R.C. Gamner, Short-Term Test Systems for Detecting

Carcinogens.

Springer Verlag Berlin, Heidelberg, New York (1980).

Page 29 of 57
Report; Project No.: 40M0437/034093

APPENDIX I

TABLES 1 -9

(STANDARD PLATE TEST)

Page 30 of 57
RAWF

Report; Project No.: 40M0437/034093

BASF hActteng-eleulanbft 01

Experienta.l Toxicology and Ecology

STUDY DIR~ECT~OR: DR. DTGELNAPDT

PROJECT NO. 40H0437/034093

SUBSTANcz NAME: Alkali Blue Dry 6100 /Lot 2236309

mSUBTAqCE 140. 03/0437-3

TESTDATB 06.=OV.2003

METHOD :STANDARD PLATE TEST

VOLUME 100 ul/plate

STRAI TIA 1535

VEHICLE :DM80

:NTHOUT WIm S-9 N= WITH S-9 MIX (1:9)

DOSE/PLATE RX" X SD PAC REV X SD EEC TITRE

DIw0 16 17 3 1.0 19 16 3 1.0 25

21 14 24

is 16 22

23 ug 17 21 4 1.2 16 14 3 0.8

25 14

20 11

115 ug 16p 21 6 1.2 16P 15 2 0.9

28P 12P

19P) 16P

575 ugI 21P 18 3 1.0 14P 17 5 1.0

15P .22P

lop 14P

2 875 ug 33/P 3 2 0.2 03/PI

53/p 03/P 4

2E/p 03/P 3

5 750 ug 03/P . 0/P 0

03/P 03/P 0

03/P 03/P 0

meb 5.0 ug 936 921 58 53.1

857

970

2-A?. 2.5 ug 170 151 21 9.2

154

128

(BE-revertantf/plate, N=ineai, SD-standard deviation, EEC>-factor)

P: PMECIPITALTION

B: MRDUCEO BACKGR0UND GROWTH

Page 31 of 57
i -- It

BASF

Report; Project No.: 40M0437/034093

RM Aktiengesel I dcaft 02

Experimental Toxicology and Ecology

STUDY DIPSCTO1R: DR. ENGELHARDT

PR0OJ3C7 IM. :40M&0437/034093

SUBSTANCE lMSE: Alkali Blue Dry 62100 /Lot 2236300

suBsTANC no. 03/0437-3

TESTDA22 06.2W.2003

METHOD 87AIIDAD PLATE TEST

VOLM : 100 ul/plate

STEAME : VA 100

S9-I WITHUT B-9 1= ITo 5-9 MIX1 (1: 9)

D3EPAI 4V M4 SD 11= P" m SD PAC TIME

INO104 110 7 1.0 109 118 8 1.0 32

110 121 28

117 124 28

23 ug 113 107 6 1.0 113 107 5 0.9

106 105

101 103

115 ug 100P 104 5 0.9 103P 103 2 0.9

1031 1051

1091 1011

575 ug 901 95 5 0.9 891 91 3 0.8

991 941

97P 901

2 875 ug 03/1 M1/1 11 4 0.1 1

O3/p 7B/P 0

03/1 143/P 0

5 750 mg 03/P 03/P 0

03/1 OB/p 0

0831 03/p 0

MR=U 5. 0 ug 970 969 64 9.8

1031

904

2-AA 2.5 ug 953 917 36 7.8

917

882

(BEV-revertants/plate, Mmmmu, SD-standard daviation, YAC-fantor)

1: PRECIIIWv

B: REDUE RXCKG~tOUND GRWTH

Page 32 of 57
-- -~BASF

Report; Project No.: 40M04371034093

RAST AktiemlgefllIBChaft 03

Experjmnta. Toxicology and Ecology SUYDPZTR R NEHM

PRO.JECT NO. :40M0437/034093

SUBsTA~Cz NMM: Alkali Ithe Dry 6100 /Lot 223 6308

,SUBSTANCE No. :03/0437-3

TBBTDATE 06.U0v.2003

METHOD STANDARD PLATE TEST

VOW)UM 100 ul/plate

sTRAN : TL 1537

VEHICLE DM80

s9-NIX WITHOUT S-9 M= WITH 8-9 MIX (1:9)

DOSX/PLATE REV If SD FAC ?"V M SD PAC T1TER

DM80 14 12 2 1.0 10 11 1 1.0 28

12 11 17

10 12 23

23 ug 6 a 3 0.7 7 11 4 1.0

7 15

11 10

=15 ug 12P 11 2 0.9 lop 10 1 0.9

sP lop

12P lip

575 12g 6P 5 2 0.4 13P 9 5 0.8

3P 4P

6?P lip

2 875 ug 03/P 33/P 2 1 0.2 0

03/P 13/P 0

03/P 23/P 0

5 750 ug 03/P 03/P 0

03/P 03/P a

03/P CE/P 0

Axc 100 ug 460 425 32 35.4

419

396

2-A 2.5 ug 147 149 11 13.5

161

139

(pEv-revertants/plate, Mnmn, SD=tandard deviat-ion, yAC-factor)

P: PPECIPITATION

B: P.EWMME 3P.CK0OtND GROM

Page 33 of 57
BASF

Report; Project No.: 40M0437/034093

BA81 hkjtjeng~eelahwft 04

zxpermumntal Toioology and Ecology

STUDY DIRECTOR: DR. ENGELHMIT

PRO.)EC NO. 40W1437/034093

8StXnN kqA): Alka-i E1lie Dry 6100 /Lot 2236308

8U3STAEC No. :03/0437-3

TEBTDAU : 06.2WV.2003

HETBOD : TAiNMRD Pla= E ST

VOLUM :100 ul/plate

87PJ13F TA 98

89-m= : VTOUT S-9 3= Tm S-9 xixX (1:9)

DOSE/PlATE F"V X4 ED PAC REV K4 SD FAC TITE

DM80 38 33 5 1.0 38 38 4 1.0 26

31 41 34

29 34 31

23 wg 27 27 3 0.8 28 40 14 1.1

30 36

25 55

115 uq 36P 30 6 0.9 31P 30 2 0.8

25P 27?

28? 311'

575 ug 34P 27 7 0.8 26P 29 6 0.8

28? 24?

20? 36P

2 875 wg 03/P 1OB/P 9 5 0.2 0

03/P 43/P 0

03/P BP 0

5 750 ug 03/P . 0/P0

03/P oB/D 0

wiow 10 ug 740 740 53 22.7

793

688

2-Ak 2.5 ug 900 877 23 23.3

854

878

(XWV-evertanta/plate, )i-ean, SD-standard deavlation, FAC-factor)

P: P3ECIPI2TIMC

3: 3RDMCED BACKGR0UND GROTH

Page 34 of 57
BASF

Report; Project No.: 40M04371034093

EASE AktjeiezuefLschaft 05

Experimental Toxicology anid Ecology SUYDRZ0:D.EGLAD

PRO3OCT NO.- 4014437/034093

SM3SMAV MU: Alkali RLue Dry 6100 /Lot 2236308

SMTAMU No. 03/0437-3

TESTI)A22 06.NOV.2003

METHOD STAN~DARD PLATE TEST

VOLUME 100 ul/plate

STIULIV : .col-t H'2 uvrA

VEHICLIE IW

59JLX WTOUT S-9 MIX WITH S-9 k=X (1: 9)

z)OSE/PLI.Tg REV x SD FAC PXV M SD EAC TITRE

- DM80 30 31. 1 1.0 29 34 5 1.0 37

30 36 35

32 37 44

23 Ug 31 30 6 1.0 25 32 6 0.9

36 33

24 37

1-5 g 35P 32 3 1.0 40P 30 9 0.9

30P 22P

31P? 27P

575 ug' 26? 25 1 0.a 21P 25 4 0.7

25P 26?

24? 28P

29875 U9 22? 19 4 0.6 21? 18 3 0.5 26

14? 27P 22

20? 15? is

5 750 ug lip 15.4 0.5 12P 14 3 0.4 12

19? 17P 17

14P 13P 11

4-N100 5.0 ug 813 754 57 24.6

748

700

2-A7A 60 ug 200 214 12 6.3

217

224

(REVrevetants/plate, Itean, SD-standaxd deviatiori, PAf-factor)

P: PEIRITATIK

Page 35 of 57
iwrni Wu UJJ'U1 1r1 Un IBASF

Report; Project No.: 40M0437/034093

BASF Aktiengesellseliaft 06

zxperimental. Toxicology and Ecology

STUDY DIP2CTOR: DR. E2NQEHA1MT

PRD3=C NO.- 40IM437/034093

suB8TA1NE was: Alkali Blue Dry 6100 /Lot 2236308

sDBSTA1vE No. 03/0437-3

TESTDP.TH 04.DEC.2003

14ETHOD STBNAM PLATE TEST

VOLtMM 100 ul/plate

STRLAN TP. 153

VEHICIS DM8=0

89-wIX :WITHOUT S-9 NI WTH 6-9 NIX (1: 9)

DOSE/PlATE REV K SD FAC AMV b SD PAC TITEE

DKO16 16 3 1.0 14 16 3 1.0 36

is 20 30

13 i5 30

0.8 u 16 17 1 1.1 16 16 4 1.0

is 20

1s 13

4 ug 11 14 3 0.9 13 15 3 0.9

13 18

17 13

20 ug 22 17 5 1.1 14 13 2 o.e

16 11

12 1.4

100 ug 113? 19 7 1.2 171' 16 2 1.0 22

21P 141' 31

24P' 17P' 27

500 ug 141' 13 .1 0.8 141' 15 2 0.9 16

121' 171' 17

1.33? 13' 21

low 5. 0 ug 1041 1050 17 67.0

1070

1039

2-AIL 2.5 uq 169 163 12 10.0

150

171

(PRvrevertants/plate, M3mam, SDlstardard deviation, FAC-factor)

1': PRECIPITATIN

Page 36 of 57
BASF

Report; Project No.: 40M0471034093

BAsF 3itiengenellschaft 07

xpei~mtai Toxicology and Ecology

STUDY DIRECTOR: DR. ZffGMLEABDT

R0NC NO. 4010437/034093

StJBSTAM m=N: Alkali Blue Dry 6100 /lot 2236308

sUBSTAwcE No. 03/0437-3

TEsTDA : 04.DEC.2003

METHOD :STANDAR PLATE TEST

VOLUme 100 12/plate

STPam : TAt 100

VEHICZE : DMSO

39-MIX : WITHOUT S-9 B33 WITH 8-9 lax (1: 9)

DOSE/PLATE REV7 x SD PAC riEV X SD PAC TITAR

- nSO, 140 1-20 18 1.0 114 12.0 4 1.0 24

107 109 27

1.13 107 38

0.8 ug 104 105 3 0.9 107 112 6 1.0

103 110

109 119

4 ug 113 107 7 0.9 102 105 7 1.0

108 100

100 113

20 ug 103 107 4 0.9 113 114 2 1.0

2.11 117

107 113

100 ug 120P 112 9 0.9 102P 105 5 1.0 17

114P ilOP 27

103P 102P 26

500 ug loop 92 7 0.8 uop 104 5 0.9 18

90p 102P 20

87P 100p 17

bRO 5.0 ug 1131 1084 43 9.0

1073

1047

2-hP. 2.5 vq 900 951 45 8.6

983

970

(pzv-revertants/plate, YAmen SD=standaxd devIation, FAC-factor)

1: pRSCIVITEXIO

Page 37 of 57
RASP

Report; Project No.: 40M0437/034093

3181 hkt~eng~eloshaft 08

Experimental Toxiocogy and Ecology

PROJCT :4OW47/03093STUDY N. DIRlECTOR: DR. ENGEL~IEDT

SUBESTANCE NAME: Alkali Rlue DrY 6100 /Lot 2236308

SUBS7XM~CZ NO. 03/0437-3

TzsTnagy 04.DZC.2003

METHIW : MT.AD PLATE 7r6ST

VOIXWE :100 ul/plate

STREAN TA 1537

VEHIcLE DMtw0

S9= :WITHOUT 8-9 WITHfl 8-9 M=X (1:9)

DOSE/PlATE MEV K SD PAC RE" x SD NrC TITE DXS0 10 11 2 1.0 15 1.1 4 1.0 31 13 8 29 9 10 28

O.8 ug 14 10 5 0.9 9 7 2 0.6 5 6

10 6

4 uq 7 6 1 0.5 8 12 4 1.1

5 14

5 15

20 ug 10 a 2 0.7 11 n1 0 1.0 6 1

7 1

100 ug 73W 7 1. 0.7 9W 10 2 0.9 24 6P 1"W 27 sW 9p 21.

500 ug 2W 4 .2 0.4 7P 7 4 0.7 26 6P 11P 27

5P 4p 25

AA.C 100 ug 436 411 24 38.6

388

410

2-AlL 2.5 ug 144 134 9 12.2

130

127

(REV-revertants/plate, Mamean, sD-standard deviation, pAC-factor)

Page 38 of 57
FLI ~ 'n~n

BASF

Report; Project No.: 40M0437/034093

BAST )ktiexqfeellaft 09

Expex-imntal Toxicology and Rcology

STUDY D3hECTOR: DR~. ENGMHAPDT

PROJIECT NO. :40M0437/034093

SS3Txm~ MWd: ALkaUi Blue Dry 6100 /Lot 2236308

sBTSmCR No. :03/0437-3

TESIDAE 04 .DHC. 2003

IdETBODST~MAW PLATE TEST

VOLUME 100 ill/plate

STPJLF : T 98

VEICLE : Dwso

59-= WI1THOUT S-9 W= WTH B-9 2=X (1:9)

DOSE/PZLTE FE" m SD rAc P" SD PAC TITW.E

- DamS 28 29 2 1.0 44 42 3 1.0 24

31 39 28

28 43 34

0. ug 26 30 7 1.0 46 39 6 0.9

38 34

27 37

4 ug 33 28 6 1.0 31 29 2 0.7

30 27

22 30

20 ug 30 27 4 0.9 33 36 3 0.9

22 37

28 39

100 ug 19W 21 4 0.7 42P 35 6 0.8 27

262 30P 24

19P 32W 31

500 ug 16P 20 4 0.7 43P 39 8 0.9 21

23P 44W 28

22Y 30P 24

NOPD 10 ug 602 605 25 20.9

631

582

2-xA 2.5 ug 770 718 47 17.1

681

702

(pEv-revertants/plate, M~mean, SD=standard deviation, FA-factor)

P: PiEclPITATWE

Page 39 of 57
BASF

Report; Project No.: 40M0437/034093

APPENDIX 2

TABLESI10-14

(PREINCUBATION TEST)

Page 40 of 57
BARF

Report; Project No.: 40M0437/034093

BABE Jkkttengeael SCAMat 10

Exper±mntal Toxicology and Ecology

STUDY DIPZCCTOR: DR. ERGELHAMT2

P1RO33C N0. 4014437/034093

s1J3STAWIE 2AME: Alkali Blue Dry 6100 /Lot 2236308

sU38TMAc No. 03/0437-3

TESTDAR I0.DEC.2003

dE'THOIC PF23NCUSATION TEST

VOLUME 100 ul/plate

STRA" : T 1535

VEHICLX DM80

S9-f~ : WITHOUT S-9 MTT WTH 5-9 MIX (1: 9)

DOSE/PLATE F"V 1 SD XAC REV x SD EA= TITE

DM80 18 17 2 1.0 19 17 3 1.0 27

19 13 30

15 1s 23

4 sqg 15 16 2 0.9 12 15 3 0.9

14 17

18 16

20 ug 22 16 5 0.9 11 14 3 0.8

14 16

13 15

100 sq Isp 16 1 0.9 16P 1.5 1 0.9

162 14P

ISP 5

500 ug 13P 12 2 0.7 12P 14 2 0.8 17

lop 16P 1s

14P 14P 22

1 500 sq 12BhP 10 2 0.6 83/P 9 1 0.5 20

93/P 103/P 11

83/P 93/P 16

NNWa 5.0 sq 643 644 14 37.1

630

658

2-hA 2.5 sq 100 105 6 6.3

11

103

(EEV.=evertant/plate, Vmuean, SD-.tandard deviation, YAO-factor)

P: PRCM7IC2IG

3: P.EcD BAC3PR0UU GROTH

Page 41 of 57
BAUSF

Report; Project No.: 40M0437/034093

IL"AR.kit-iengesllahaf t

Eprimntal Toxicmoog and Ec-ology

STUDY DIRECTOR: DR. M!SGfLN)T

PROJECT no.- 40W437/034093

suWsAcz NME: A13cali BI. Dry 6100 /Lot 2236308

8tJBSTRNOE NO. 03/0437-3

TESTDA3Z 1O.DZC.2003

METHOD PRZINCMLTX~fI TEST

VOLUME 100 ni/plate

BSPAXN : 72L 100

VEHICLE DhM0

89-= WXTHOUT 8-9 2= wTH 8-9 MIX (1:9)

DOSE/ITE REv x SD FAC REv X SD MC TIuhE

DmS0 110 107 4 1.0 103 109 5 1.0 24

102 111 29

108 113 27

4 ug 102 105 a 1. 0 1.02 108 7 1.0

98 1125

114 107

20 ug 103 162 2 1.0 104 105 4 1.0

104 102

100 109

100 ug 120? 103 15 1.0 loop 100 2 0.9

95? 1013?

93? 982,

500 ug 98?, 87 10 0.8 95? 93 3 0.9 22

823P 90P 23

SOP 93P 27

1 500 ug 40R/P 37 .3 0.4 64B/p 63 13 0.6 11

37B/P 50R/P 17

353/P 753/p 16

NIgM 5.0 ug 632 664 29 6.2

672

688

2-uLA 2.5 ug 599 619 21 5.7

641

617

(p3v-=evextants/plate, X-mn SDstaudaxd deviation, Plc-factor)

P: PmaEI2W(TIQ

3: PmuXJw BID8ROVUND WRTH2

Page 42 of 57
BASF

Report; Project No.: 40M0437/034093

Ex"Y Aktiengesellschaft 12

Experimental ToxLcology and Ecology

STUDY DIhZCTCR: DR. EVGRLSAIRDT

POJECT NO. 40M0437/034093

smjsT1Icg iNE: Alkali Blue Dry 6100 /Lot 2236308

sUssTAwcz no. 03/0437-3

TEBTDATZ 10.DEC.2003

METHOD : PEIrNCORATXCE TEST

VOLUME :100 ul/plate

STPA3N :TA 1537

VEHICLE :DM80

S9-m= : WITHOUJT S-9 NE= WITH S-9 M= (1:9)

D0SE/PLATE AE" X SD YAC p"V w SD JAC TTE

DKSO 12 10 2 1.0 9 9 1 1.0 26

9 9 23

10 a 25

4 ug 14 10 3 1.0 8 7 4 0.8

8 11

9 3

20 ug 10 8 2 0.8 a 8 0 0.9

78

100 ug 4p 4 2 0.4 7P 6 2 0.7

6p 7p

2P 4P

500 ug SP 5 2 0.5 6p 6 1 0.7 24

3P 6p 21

7P 7P 18

1 500 iag 23/p 2 1l 0.2 6B/P 4 2 0.5 15

3B/P 3B/9 1.2

3B/2 3B/P 17

AMc 100 ug 439 446 50 43.2

500

400

2-hit 2.5 ug 103 99 7 1.1.4

91

103

(R-revrtant/plate, ld-mean, D-standard deviation, PAC-factor)

P: FRECIpIwkTIOK

B: PED~ BACNROUN GROTH

Page 43 of 57
Report;, Project No.: 40M04371034093

iWAc)tj..nesllschiaft. 13

prxperiueftal. Toxicology and Ecology

STUDY DIRZCTOR: DR. ENRIZLUMT

pmrOOZC No. 40H0437/034093

STJBBTANC NANE Alkali Blue Dry 6100 /Lot 2236308

SMBTANE Iwo. :03/0437-3

TEMMAS 10.MEC.2003

METBOD : PEDCM~ATIH TAB?

VOL= : 100 Wolate

ST2lIN TA 98

UWmCLR DWEO

09-3=X WTNOUI' S-9 J= WITHS8-9 baX (1:9)

DOSE/PLAE I"V m SD FAC REV X SD PAC TITE

UNSO 19 25 6 1.0 33 31 3 1.0 27

28 28 35

29 32 32

4 wg 30 29 5 1.2 32 29 2 0.9

34 28

24 28

20 uO 20 23 3 0.9 30 30 3 1.0

25 33

24 27

100 ug 19P 21 4 0.8 26P 27 2 0.9

26P 29P

isp 26P

500 ug 16P 17 2 0.7 30p 29 2 0.9 10

15w 27P 10

19W 30P 15

1500o ug 2S/P 3 .2 0.1 203/P 18 3 0.6 10

23/P 143/P 11

63/P 192/p 15

NOpjD 10 wg 870 885 22 34.9

911

875

2-lA 2.5 ug 604 563 46 18.2

571

513

(RMv-revertants/plate, U-ne=x, SD-standard deviatioz, rAC=factor)

P: pE3CIpITA2'IK

3: R1DUoE BACPMROD GP.O1T

Page 44 of 57
BASF

Report; Project No.: 40M0437/034093

BASF Aktieng'esellschaft 14

Eaqmei-vental, Toxicology and Ecology, STUDY DIREVCTOR: DR. EWGRJIARr)T

WRO17ZC NO. :40M0437/034093

slUSTA=c HAW: Alkali. Blue Dry 6100 /Lot 2236308

sMLSTABcZ No. :03/0437-3

7RSTDA10 I.DEC. 2003

METHOD WEIN1CMULTIOT TEST

VOLO1ME :100 ni/plate

STP~uR : .col± V122 uvxcA

VEHICLE : M

89-MIX :W7THOUT S-9 MX WITH S-9 lax (1: 9)

DOSE/PXATE RE" w SD 3= REV X SD FE5C TITER

- DNBO 30 31 3 1.0 32 32 3 1.0 35

28 29 41

34 34 33

4 ugr 26 31 5 1.0 28 30 5 1.0

34 36

34 27

20 ug 21 27 6 0.9 25 27 2 0.9

28 27

32 29

100 ug 301? 28 2 0.9 302 24 5 0.8

26P 202

28P 22P

500 ug 223? 21 1 0.7 27P 23 5 0.7 26

20P 25P 27

21P lap 22

1 500 ug 24B/P 16 . 7 0.5 123/P 13 2 0.4 15

123/P 14B/P 14

14B/P 15B/P 19

4-NQO 5.0 ug 672 658 31 21.5

622

680

2-AA 60 U9 200 206 6 6.5

207

232

(p~v.revrtants/plate, H-ckan, SD-standard deviationi, rhO-factor)

B: PZDtJCED BAOKSROMID GROWTH

Page 45 of 57
_____~~- -l 1 II It11~UJu

BALSF

Report; Project No.: 40M0437/034093

APPENDIX 3

STERILITY CONTROL

Page 46 of 57
BAS=

Report; Project No.: 40M04371034093

STERILITY CONTROL

1st Experiment

" 2.0 ml soft agar 

No growth

0.5 ml S-9 mix No growth

0.5 ml buffer No growth

0.5 ml vehicle No growth

5 750 jig test substance No growth

2nd Experiment

0.5 ml vehicle No growth

500 pg test substance No growth

3rd Experiment

2.0 ml soft agar No growth

0.5 ml S-9 mix No growth

0.5 ml buffer No growth

0.5 ml vehicle No growth

1 500 pg test substance No growth

Page 47 of 57
~LL~EI2 I UIU 1 1~I~1 II~rII:II~~ill'1 L I I L -1i

BASP

Report; Project No.: 40M0437/034093

APPENDIX 4

S-9 FRACTION CHARACTERIZATION

Page 48 of 57
Report; Project No.: 40M0437/034093

S-9 fraction

Preparation of S-9 fraction: June 17, 2003

Content of protein (mean*): 29.0 gI

Characterization of the S-9 mix with benzo~ajpyrene: July 14, 2003

METHOD: Standard Plate Test

S-9 FRACTIONICOFACTORS: 1:9

STRAIN: TA 100 STRAIN: TA 98

DOSEIPLATE M REV* SD- FAC* DOSEIPLATE M REV* SD* FAG'

DMSO 113.7 9.0 1.0 DMSO 34.0 3.0 1.0

0.6 Pg 258.9 32.5 2.3 0.6 pg 87.6 16.8 2.6

- 1.25 pg 368.8 26.8 3.2 1.25 pg 486.8 39.1 5.5

2.5 pg 497.5 69.3 4.4 2.5 pg 352.8 29.1 10.4

5.0 pg 639.4 29.5 5.6 5.0 Pg 492.6 46.4 14.5

(REV = revertants/plate, M = mean.SD standard deviation, FAC =factor)

=pool 1-4

Page 49 of 57
BASF

Report; Project No.: 40M0437/034093

APPENDIX 5

HISTORICAL NEGATIVE CONTROL DATA

Page 50 of 57
411111111 I No

BASF

Report; Project No.: 40M0437/034093

Historical Negative Control Data

Strain: TA 1535

Methd mi NoNo. S9 of

Mehd SBmx Ngative control 'Period Plates Min*' Mak' Mean* So

SiPr Water 03.98 - 09.03 400 11 25 18 2

SPT -DMSO 03.00 -09.03 400 11 25 18 2

SPT -Acetone 01.96- 05.03 208 11 25 19 2

SPT 1 :9 Water 03.98 - 09.03 400 12 25 18 2

SPT -1:9 DMSO 03.00 -09.03 400 10 24 18 2

SPT -1 :9 Acetone 01.96- 05.03 210 11 25 19 2

No. of

Method S-9 mix Negative control Period Plates MIi'* Mae' Mean* SID

PI Water 08.97- 06.03 400 13 25 19 2

PIT -DMSO 06-99 -07.03 400 11 25 18 2

PIT Acetone 01.96 -05.03 168 10 25 ,19 2

_____ 1 :9 Water 04.97 -06.03 400 11 25 18 2

PIT 1 :9 DMSO 05.99- 09.03 400 10 23 17 2

PIT 1 :9 I Acetone 01.96- 05.03 159 10 -25 18 2

Strain: TA 100

No. of

Method S-9 mix Negative control Period plates Ae Max* Mean* So

SPT -Water 05.98 -09.03 400 82 '155 117 14

SPT DMSO 06.00 -09.03 400 91 160 110 10

SPT - Acetone 01.96- 05.03 258 91 160 118 1s

SPT 1:9 Water 06.98--09.03 400 94 156 123 16

SPT 1:9 DMS0 08.00 -09.03 400 92 154 112 10

ISPT 1:9 Acetone 01.96- 0503 253 85. 150 -120 16

No. of

Method S-9 mix Negative control Period _ plates Min*' Max* Mean' SID

PIT -Water 06.97 - 06.03 400 90 160 117 14

PIT -DMS0 06.99- 09.03 400 64 150 112 12

PIT -Acetone 01.96- 05.03 172 91 160 1117 16

PIT 1:9 Water 05.97 -06.03 400 92 159 122 15

PIT 1:9 DMSO 06.99 -09.03 400 80 159 112 13

PIT 1: 9 Acetone 01.96- 05.03 156 96 160 1119 1

* a revertants/plate PIT = preincubation test

SPT = standard plate test SID - standard deviation

Page 51 of 57
Report; Project No.: 40M0437/034093

Historical Negative Control Data

Strain: TA 1537

No. of

Method S-9 mix Negative control Period plates- Min* Max* Mean*. SID

SPT -Water 03.9 - 09.03 400 6 16 10 2

SPT -DMS0 03.00 -09.03 400 5 16 10 2

SPT - Acetone 01.96 -05.03 213 5 18 10 12

SPT I :9 Water 03.98 -09.03 400 6 19 11 2

SPT I:9 DMSO 03.00 - 09.03 400 5 19 11 2

I SPT I 1:9 Acetone 01.96 -05.03 213 7 20 12 12

No. of

Method S-9 mix Negative control Period plates Min' Max* Mean' SID

PIT -Water 05.97 -06.03 400 5 19 10 2

PIT -DM80 06.99 - 09.03 400 5 17 10 2

PT- Acetone 01.96 -05.03 168 6 17 10 2

PIT 1 :9 Water 04.97 - 06.03 400 6 20 11 2

PIT 1:9 DM80 04.99 -09.03 400 5 16 10 2

PIT 1 :9 Acetone 01.96- 05.03 162 15 20 11 2

Strain: TA 98

No.of

Method S-9 mix Negative control Period 6lha t e. Min' Max* Mean' SID

SPT -Water 06.98 - 09.03 400 17 44 28 5

SPT -DM80 07.00 - 09.03 400 20 45 29 5

SPT -Acetone 01.96 -05.03 215 19 50 29 5

SPT 1:9 Water 06.98 - 09.03 400 20 49 36 6

SPT 1 :9 DMSO 08.00 - 09.03 400 20 50 36 6

SPT 1 :9 Acetone 01.96 - 05.03 214 26 50_ 38 -5

'No. of

Method S-9 mix Negative control Period viates Mm'0* Max* Mean' SID

PIT -Water 05.97 - 06.03 400 18 44 27 4

PIT -DMSO 06.99 - 09.03 1400 17 40 27 4

PIT -Acetone 01.96 -05.03 168 19 145 28 5

PIT I:9 Water 04.97 - 06.03 400 17 50 36 6

PIT 1 :9 DMSO 06.99 - 09.03 400 20 49 32 5

PIT I:9 1 Acetone 01.96 -05.03 161 20 50 37 6

= revertants/plate PIT = prelncubation test

SPT =standard plate test SID = standard deviation

Page 52 of 57
BtAS

Report; Project No.: 40M0437/034093

Historical Negative Control Data

Strain: W132 uvrA

No. of

Method S__9_mix Negative control Period plates Min* Max' Mean* SID

SPT -Water 11.97.-09.03 400 20 40 31 5

SPT -DMSO 01.00.-09.03 400 22 50 32 5

SPT -Acetone 01.96 -05.03 200 18 150 33 6

SPT 1 :9 Water 01.98 -09.03 400 20 49 35 6

SPT 1 :9 DMSO 01.00 -09.03 400 23 50. 35 6J

SPT 1 :9 Acetone 01.96.05.03 200 115 50 37 71

No. of

Method S-9 MIX Negative control Period plates Min* MaX* Mean* SID

PIT -Water 04.97.-06.03 400 21 50 31 5

F PIT -DMS0 07.99 -09.03 400 19 49 32 -5

PIT -Acetone 01.96.-05.03 -157 20 47 31 5

PIT 1 :9 Water 01.97 -06.03 400 23 50 37 6

PIT 1 :9 DM50 06.99 -09.03 400 19 50 32 6

PIT 1 :9 Acetone 01.96 -05.03 157 20 40 33 6

= revertantslplate PIT =preincubation test

SPT = standard plate test SD m standard deviation

Page 53 of 57
RASF

Report; Project No.: 40M0437/034093

APPENDIX 6

HISTORICAL POSITIVE CONTROL DATA

Page 54 of 57
BASF

Report; Project No.: 40M0437/034093

Historical Positive Control Data

Strain: TA 1535

IPositive control No. of

Meho S- mi Peid pae-i*Mx o*S -ip 

SPT - MNNG 5.0 02.01 - 09.03 400 500 1649 776 199o

S PT 1 :9 2-AA 2.5 02.01 -09.03 400 85 325 146 38J

SPos ive control No. of I

Method S-9 MIX PglplI Period plates Min* Max* Mean* SD

PIT - MNNG 5.0 07.00 -09.03 400 502 1357 785 150

PIT 1 :9 2-AA 2.59 06.00 -09.03 400 so 345 129 31

Strain: TA 100

I 1 Positive control INo. ofII

Method S-9 mix Pglpl Period .1plates* #An* Max* ~vrS

SPT I - I MNNG 5.0 I03.01 -09.03 J400 I501 I1747 871 262j

SPT 1:9- _2-AA 2.5- 03.01 -09.03 )400 507 1708 859 215

Posiive ontrl -N. o

Method S-9 mix ipglpi Period plates Min*MaW't* Mean'f SD

PIT- NNG 5.0 07.00 -09.03 400 502 1583 Is 188

PT I :9 2-AA 2.5 06.00 - 09.03 400 500 1125 I675 135

= revertants/plate

SPT = standard plate test

PIT =prelncubatlon test

SD = standard deviation

2 - AA =2-aminoanthracene

MNNG N-methyl-N-ntro-N-nltrosoguanidine

Page 55 of 57
BASF

Report; Project No.: 40M0437/034093

Historical Positive Control Data

Strain: TA 1537

Positive control No. of IMethod S-9 mix PglplI Period Min* Max* Mean* SD

S I AAC 100 301.01-09.031 400 300)1147 443 1041

SPT I :9 2-AA 2.5 J01.01 - 09.03 400 71 j200 127 2

I IPositive conitrol No. ofI I

Method S-9 mix P. 9p Period plates Min* Max* Mean* l SDJ

PIT I - AAC 100 07.00 -09.03 400_ 278 8851435 I9 3

PIT 1:9 2-AA 2.5 06.00 -09.03 400__ 50 200 I112 I20

Strain: TA 98

II 1 ~Positive controlN. f

.- Method' S-59 MIX pa/pl atrio MW Max* M#ean' SD IISPT j - I NOPD 10 02.1 09.03 00 34 120 737 132

SPT 1:9 2-AA 2.5 0301-09.03 00 504 138 699 139

Positive control 1No, ofI

Method i5-9 mix , Ip Period_ plates Mi*MaX' Mean* SD

PIT - NOPI1 07.00 -09.03 I400 39 1129 ~.735 135

PIT 1:9 2-AA 2.5 06.00 -09.03 400 1 5621985 90

=revertafltsplnte

SPT = standard plate test

PIT =proincubatlon test

sD w standard deviation

2- AA = 2-aminoanthracene

AAC =9-aminaacridine

NOPD 4-nitro-o-phenylendiamine

Page 56 of 57
BASF

Report; Project No.: 40M0437/034093

Historical Positive Control Data

Strain: WP2 uvrA

I I IPositive control No. of

Method S-9 mix gjgIpl Period plates Min* MWx Mean* SD fiSPT - I4-NQO 5.0 12-00 -09.03 400 500 1620 I676 171

ISPT I:9. 2-AA 60.0 01.01 - 09.03 400 155 476 244 -40

I Positive control

Method S-9 mix jlig/pl ___________

PIT 4-N 00 5.0 02.00 - 09.03 400 500 995 588 76

PIT 1 9 2-AA 60.0 06.00 - 09.03 400 170 384 232 26

arevertanits/plate

SPT = standard plate test

PIT a preincubation test

SD = standard deviation

2 - AA = 2-aminoanthracene

4-NQO m 4-Nitroquinoline-N-oxide

Page 57 of 57